Imaginal Discs Research Articles

A peptide derived from TID1S rescues frataxin deficiency and mitochondrial defects in FRDA cellular models.

Friedreich's ataxia (FRDA), the most common recessive inherited ataxia, results from homozygous guanine-adenine-adenine (GAA) repeat expansions in intron 1 of the FXN gene, which leads to the deficiency of frataxin, a mitochondrial protein essential for iron-sulphur cluster synthesis. The study of frataxin protein regulation might yield new approaches for FRDA treatment. Here, we report tumorous imaginal disc 1 (TID1), a mitochondrial J-protein cochaperone, as a binding partner of frataxin that negatively controls frataxin protein levels. TID1 interacts with frataxin both in vivo in mouse cortex and in vitro in cortical neurons. Acute and subacute depletion of frataxin using RNA interference markedly increases TID1 protein levels in multiple cell types. In addition, TID1 overexpression significantly increases frataxin precursor but decreases intermediate and mature frataxin levels in HEK293 cells. In primary cultured human skin fibroblasts, overexpression of TID1S results in decreased levels of mature frataxin and increased fragmentation of mitochondria. This effect is mediated by the last 6 amino acids of TID1S as a peptide made from this sequence rescues frataxin deficiency and mitochondrial defects in FRDA patient-derived cells. Our findings show that TID1 negatively modulates frataxin levels, and thereby suggests a novel therapeutic target for treating FRDA.

Characterisation of the in-vivo miRNA landscape in Drosophila ribonuclease mutants reveals Pacman-mediated regulation of the highly conserved let-7 cluster during apoptotic processes.

The control of gene expression is a fundamental process essential for correct development and to maintain homeostasis. Many post-transcriptional mechanisms exist to maintain the correct levels of each RNA transcript within the cell. Controlled and targeted cytoplasmic RNA degradation is one such mechanism with the 5'-3' exoribonuclease Pacman (XRN1) and the 3'-5' exoribonuclease Dis3L2 playing crucial roles. Loss of function mutations in either Pacman or Dis3L2 have been demonstrated to result in distinct phenotypes, and both have been implicated in human disease. One mechanism by which gene expression is controlled is through the function of miRNAs which have been shown to be crucial for the control of almost all cellular processes. Although the biogenesis and mechanisms of action of miRNAs have been comprehensively studied, the mechanisms regulating their own turnover are not well understood. Here we characterise the miRNA landscape in a natural developing tissue, the Drosophila melanogaster wing imaginal disc, and assess the importance of Pacman and Dis3L2 on the abundance of miRNAs. We reveal a complex landscape of miRNA expression and show that whilst a null mutation in dis3L2 has a minimal effect on the miRNA expression profile, loss of Pacman has a profound effect with a third of all detected miRNAs demonstrating Pacman sensitivity. We also reveal a role for Pacman in regulating the highly conserved let-7 cluster (containing miR-100, let-7 and miR-125) and present a genetic model outlining a positive feedback loop regulated by Pacman which enhances our understanding of the apoptotic phenotype observed in Pacman mutants.

Automated counting of Drosophila imaginal disc cell nuclei.

Automated image quantification workflows have dramatically improved over the past decade, enriching image analysis and enhancing the ability to achieve statistical power. These analyses have proved especially useful for studies in organisms such as Drosophila melanogaster, where it is relatively simple to obtain high sample numbers for downstream analyses. However, the developing wing, an intensively utilized structure in developmental biology, has eluded efficient cell counting workflows due to its highly dense cellular population. Here, we present efficient automated cell counting workflows capable of quantifying cells in the developing wing. Our workflows can count the total number of cells or count cells in clones labeled with a fluorescent nuclear marker in imaginal discs. Moreover, by training a machine-learning algorithm we have developed a workflow capable of segmenting and counting twin-spot labeled nuclei, a challenging problem requiring distinguishing heterozygous and homozygous cells in a background of regionally varying intensity. Our workflows could potentially be applied to any tissue with high cellular density, as they are structure-agnostic, and only require a nuclear label to segment and count cells.

A mismatch in the expression of cell surface molecules induces tissue-intrinsic defense against aberrant cells

Tissue-intrinsic error correction enables epithelial cells to detect abnormal neighboring cells and facilitate their removal from the tissue. One of these pathways, "interface surveillance," is triggered by cells with aberrant developmental and cell-fate-patterning pathways. It remains unknown which molecular mechanisms provide cells with the ability to compare fate between neighboring cells. We demonstrate that Drosophila imaginal discs express an array of cell surface molecules previously implicated in neuronal axon guidance processes. They include members of the Robo, Teneurin, Ephrin, Toll-like, or atypical cadherin families. Importantly, a mismatch in expression levels of these cell surface molecules between adjacent cells is sufficient to induce interface surveillance, indicating that differences in expression levels between neighboring cells, rather than their absolute expression levels, are crucial. Specifically, a mismatch in Robo2 and Robo3, but not Robo1, induces enrichment of actin, myosin II, and Ena/Vasp, as well as activation of JNK and apoptosis at clonal interfaces. Moreover, Robo2 can induce interface surveillance independently of its cytosolic domain and without the need for the Robo-ligand Slit. The expression of Robo2 and other cell surface molecules, such as Teneurins or the Ephrin receptor is regulated by fate-patterning pathways intrinsic and extrinsic to the wing disc, as well as by expression of oncogenic RasV12. Combined, we demonstrate that neighboring cells respond to a mismatch in surface code patterns mediated by specific transmembrane proteins and reveal a novel function for these cell surface proteins in cell fate recognition and removal of aberrant cells during development and homeostasis of epithelial tissues.

Ionizing radiation induces cells with past caspase activity that contribute to the adult organ in Drosophila and show reduced Loss of Heterozygosity

There is increasing recognition that cells may activate apoptotic caspases but not die, instead displaying various physiologically relevant consequences. Mechanisms that underlie the life-or-death decision in a cell that has activated apoptotic caspases, however, are incompletely understood. By optimizing a published reporter for past caspase activity, we were able to visualize cells that survived caspase activation specifically after exposure to ionizing radiation in Drosophila larval wing discs. We found that cells with X-ray-induced past active caspases (XPAC) did not arise at random but were born at specific locations within the developing wing imaginal discs of Drosophila larvae. Inhibiting key components of the apoptotic pathway decreased XPAC number, suggesting that apoptotic signaling is needed to induce XPAC cells. Yet, XPAC cells appeared in stereotypical patterns that did not follow the pattern of IR-induced apoptosis, suggesting additional controls at play. Functional testing identified the contribution of wingless (Drosophila Wnt1) and Ras signaling to the prevalence of XPAC cells. Furthermore, by following irradiated larvae into adulthood, we found that XPAC cells contribute to the adult wing. To address the relationship between XPAC and genome stability, we combined a reporter for past caspase activity with mwh, an adult marker for Loss of Heterozygosity (LOH). We found a lower incidence of LOH among XPAC compared to cells that did not activate the reporter for past caspase activity. In addition, at time points when wing disc cells are finishing DNA repair, XPAC cells show an anti-correlation with cells with unrepaired IR-induced double-stranded breaks. Our data suggest that non-lethal caspase activity safeguards the genome by facilitating DNA repair and reducing LOH after transient exposure to X-rays. These results identify a physiological role for non-lethal caspase activity during recovery from radiation damage.

A chromogenic diarylethene-based probe for the detection of Cu2+ in aqueous medium in Drosophila for early diagnosis of Alzheimer

A diarylethene-based probe (Z)-N'-((2-amino-5-chlorophenyl)(phenyl)methylene)-2-hydroxy benzohydrazide (KBH) has been proficiently developed and its structure has been confirmed by single crystal X-ray diffraction technique. It displays a selective and sensitive colorimetric sensing of Cu2+ ions in aqueous medium with a naked eye colour change from colourless to yellow. It exhibits a significantly low limit of detection as 1.5 nM. A plausible binding mechanism has been proposed using Job's plot, FT-IR, 1H NMR titration, HRMS and DFT studies. The chemosensor is effectively reversible and reusable with EDTA. Test strip kit and real water sample analysis have been shown to establish its practical applicability. Further, the potential of KBH for the early diagnosis of Cu2+ ion-induced amyloid toxicity has been investigated in eye imaginal disc of Alzheimer's disease model of Drosophila 3rd instar larvae. The in-vivo interaction of KBH with Cu2+ in gut tissues of Drosophila larvae establishes its sensing capability in biological system. Interestingly, the in-vivo detection of Cu2+ has been done using bright field imaging which eliminates the necessity of a fluorescent label, hence making the method highly economical.

Regenerative growth is constrained by brain tumor to ensure proper patterning in Drosophila.

Some animals respond to injury by inducing new growth to regenerate the lost structures. This regenerative growth must be carefully controlled and constrained to prevent aberrant growth and to allow correct organization of the regenerating tissue. However, the factors that restrict regenerative growth have not been identified. Using a genetic ablation system in the Drosophila wing imaginal disc, we have identified one mechanism that constrains regenerative growth, impairment of which also leads to erroneous patterning of the final appendage. Regenerating discs with reduced levels of the RNA-regulator Brain tumor (Brat) exhibit enhanced regeneration, but produce adult wings with disrupted margins that are missing extensive tracts of sensory bristles. In these mutants, aberrantly high expression of the pro-growth factor Myc and its downstream targets likely contributes to this loss of cell-fate specification. Thus, Brat constrains the expression of pro-regeneration genes and ensures that the regenerating tissue forms the proper final structure.

SetDB1 and Su(var)3-9 are essential for late stages of larval development of Drosophila melanogaster.

Methylation of H3K9 histone residue is a marker of gene silencing in eukaryotes. Three enzymes responsible for adding this modification - G9a, SetDB1/Egg, and Su(var)3-9 - are known in Drosophila. To understand how simultaneous mutations of SetDB1 and Su(var)3-9 may affect the fly development, appropriate combinations were obtained. Double mutants egg; Su(var)3-9 displayed pronounced embryonic lethality, slower larval growth and died before or during metamorphosis. Analysis of transcription in larval salivary glands and wing imaginal disks indicated that the effect of double mutation is tissue-specific. In salivary gland chromosomes, affected genes display low H3K9me2 enrichment and are rarely bound by SetDB1 or Su(var)3-9. We suppose that each of these enzymes directly or indirectly controls its own set of gene targets in different organs, and double mutation results in an imbalanced developmental program. This also indicates that SetDB1 and Su(var)3-9 may affect transcription via H3K9-independent mechanisms. Unexpectedly, in double and triple mutants, amount of di- and tri-methylated H3K9 is drastically reduced, but not completely absent. We hypothesize that this residual methylation implies the existence of additional H3K9-specific methyltransferase in Drosophila.

The central regulatory circuit in the gene network controlling the morphogenesis of Drosophila mechanoreceptors: an <i>in silico</i> analysis

The central regulatory circuit in the gene network controlling the morphogenesis of Drosophila mechanoreceptors: an <i>in silico</i> analysis

Impaired dNKAP function drives genome instability and tumorigenic growth in Drosophila epithelia.

Mutations or dysregulated expression of NF-kappaB-activating protein (NKAP) family genes have been found in human cancers. How NKAP family gene mutations promote tumor initiation and progression remains to be determined. Here, we characterized dNKAP, the Drosophila homolog of NKAP, and showed that impaired dNKAP function causes genome instability and tumorigenic growth in a Drosophila epithelial tumor model. dNKAP-knockdown wing imaginal discs exhibit tumorigenic characteristics, including tissue overgrowth, cell-invasive behavior, abnormal cell polarity, and cell adhesion defects. dNKAP knockdown causes both R-loop accumulation and DNA damage, indicating the disruption of genome integrity. Further analysis showed that dNKAP knockdown induces c-Jun N-terminal kinase (JNK)-dependent apoptosis and causes aberrant cell proliferation in distinct cell populations. Activation of the Notch and JAK/STAT signaling pathways contributes to the tumorigenic growth of dNKAP-knockdown tissues. Furthermore, JNK signaling is essential for dNKAP depletion-mediated cell invasion. Transcriptome analysis of dNKAP-knockdown tissues confirmed the misregulation of signaling pathways involved in promoting tumorigenesis and revealed abnormal regulation of metabolic pathways. dNKAP knockdown and oncogenic Ras, Notch, or Yki mutations show synergies in driving tumorigenesis, further supporting the tumor-suppressive role of dNKAP. In summary, this study demonstrates that dNKAP plays a tumor-suppressive role by preventing genome instability in Drosophila epithelia and thus provides novel insights into the roles of human NKAP family genes in tumor initiation and progression.

The activity of engrailed imaginal disc enhancers is modulated epigenetically by chromatin and autoregulation.

engrailed (en) encodes a homeodomain transcription factor crucial for the proper development of Drosophila embryos and adults. Like many developmental transcription factors, en expression is regulated by many enhancers, some of overlapping function, that drive expression in spatially and temporally restricted patterns. The en embryonic enhancers are located in discrete DNA fragments that can function correctly in small reporter transgenes. In contrast, the en imaginal disc enhancers (IDEs) do not function correctly in small reporter transgenes. En is expressed in the posterior compartment of wing imaginal discs; in contrast, small IDE-reporter transgenes are expressed mainly in the anterior compartment. We found that En binds to the IDEs and suggest that it may directly repress IDE function and modulate En expression levels. We identified two en IDEs, O and S. Deletion of either of these IDEs from a 79kb HA-en rescue transgene (HAen79) caused a loss-of-function en phenotype when the HAen79 transgene was the sole source of En. In contrast, flies with a deletion of the same IDEs from an endogenous en gene had no phenotype, suggesting a resiliency not seen in the HAen79 rescue transgene. Inserting a gypsy insulator in HAen79 between en regulatory DNA and flanking sequences strengthened the activity of HAen79, giving better function in both the ON and OFF transcriptional states. Altogether our data suggest that the en IDEs stimulate expression in the entire imaginal disc, and that the ON/OFF state is set by epigenetic memory set by the embryonic enhancers. This epigenetic regulation is similar to that of the Ultrabithorax IDEs and we suggest that the activity of late-acting enhancers in other genes may be similarly regulated.

Spt5 interacts genetically with Myc and is limiting for brain tumor growth in Drosophila.

The transcription factor SPT5 physically interacts with MYC oncoproteins and is essential for efficient transcriptional activation of MYC targets in cultured cells. Here, we use Drosophila to address the relevance of this interaction in a living organism. Spt5 displays moderate synergy with Myc in fast proliferating young imaginal disc cells. During later development, Spt5-knockdown has no detectable consequences on its own, but strongly enhances eye defects caused by Myc overexpression. Similarly, Spt5-knockdown in larval type 2 neuroblasts has only mild effects on brain development and survival of control flies, but dramatically shrinks the volumes of experimentally induced neuroblast tumors and significantly extends the lifespan of tumor-bearing animals. This beneficial effect is still observed when Spt5 is knocked down systemically and after tumor initiation, highlighting SPT5 as a potential drug target in human oncology.

Ectopic Expression of Human, Mouse, and Fruit Fly Aryl Hydrocarbon Receptor in <i>Drosophila</i> <i>melanogaster</i> Tissues

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor and its target genes play a fundamental role in detoxification, regulation of developmental processes, maintenance of homeostasis, as well as in the occurrence of oncological and autoimmune diseases and drug metabolism. The high conservatism of vertebrate AHR allowed us to study its functions in vivo using transformed Drosophila melanogaster fruit flies with human or mouse AHR gene and compare the ectopic effect of their expression with the expression of spineless gene, Drosophila AHR homologue. This work demonstrates for the first time that vertebrate AHR exhibits its functional activity in Drosophila embryogenesis, in leg imaginal discs and in somatic cells of female reproductive system in the absence of exogenous ligands.

The pleiotropic functions of Pri smORF peptides synchronize leg development regulators

The last decade witnesses the emergence of the abundant family of smORF peptides, encoded by small ORF (<100 codons), whose biological functions remain largely unexplored. Bioinformatic analyses here identify hundreds of putative smORF peptides expressed in Drosophila imaginal leg discs. Thanks to a functional screen in leg, we found smORF peptides involved in morphogenesis, including the pioneer smORF peptides Pri. Since we identified its target Ubr3 in the epidermis and pri was known to control leg development through poorly understood mechanisms, we investigated the role of Ubr3 in mediating pri function in leg. We found that pri plays several roles during leg development both in patterning and in cell survival. During larval stage, pri activates independently of Ubr3 tarsal transcriptional programs and Notch and EGFR signaling pathways, whereas at larval pupal transition, Pri peptides cooperate with Ubr3 to insure cell survival and leg morphogenesis. Our results highlight Ubr3 dependent and independent functions of Pri peptides and their pleiotropy. Moreover, we reveal that the smORF peptide family is a reservoir of overlooked developmental regulators, displaying distinct molecular functions and orchestrating leg development.

Dilp8 and its candidate receptor, Drl, are involved in the transdetermination of the Drosophila imaginal disc.

Drosophila imaginal disc cells can change their identity under stress conditions through transdetermination (TD). Research on TD can help elucidate the in vivo process of cell fate conversion. We previously showed that the overexpression of winged eye (wge) induces eye-to-wing TD in the eye disc and that an insulin-like peptide, Dilp8, is then highly expressed in the disc. Although Dilp8 is known to mediate systemic developmental delay via the Lgr3 receptor, its role in TD remains unknown. This study showed that Dilp8 is expressed in specific cells that do not express eye or wing fate markers during Wge-mediated TD and that the loss of Dilp8 impairs the process of eye-to-wing transition. Thus, Dilp8 plays a pivotal role in the cell fate conversion under wge overexpression. Furthermore, we found that instead of Lgr3, another candidate receptor, Drl, is involved in Wge-mediated TD and acts locally in the eye disc cells. We propose a model in which Dilp8-Drl signaling organizes cell fate conversion in the imaginal disc during TD.

The AMPK-like protein kinases Sik2 and Sik3 interact with Hipk and induce synergistic tumorigenesis in a Drosophila cancer model.

Homeodomain-interacting protein kinases (Hipks) regulate cell proliferation, apoptosis, and tissue development. Overexpression of Hipk in Drosophila causes tumorigenic phenotypes in larval imaginal discs. We find that depletion of Salt-inducible kinases Sik2 or Sik3 can suppress Hipk-induced overgrowth. Furthermore, co-expression of constitutively active forms of Sik2 or Sik3 with Hipk caused significant tissue hyperplasia and tissue distortion, indicating that both Sik2 and Sik3 can synergize with Hipk to promote tumorous phenotypes, accompanied by elevated dMyc, Armadillo/β-catenin, and the Yorkie target gene expanded. Larvae expressing these hyperplastic growths also display an extended larval phase, characteristic of other Drosophila tumour models. Examination of total protein levels from fly tissues showed that Hipk proteins were reduced when Siks were depleted through RNAi, suggesting that Siks may regulate Hipk protein stability and/or activity. Conversely, expression of constitutively active Siks with Hipk leads to increased Hipk protein levels. Furthermore, Hipk can interact with Sik2 and Sik3 by co-immunoprecipitation. Co-expression of both proteins leads to a mobility shift of Hipk protein, suggesting it is post-translationally modified. In summary, our research demonstrates a novel function of Siks in synergizing with Hipk to promote tumour growth.

Colorimetric Synchronization of Drosophila Larvae.

The rapid succession of events during development poses an inherent challenge to achieve precise synchronization required for rigorous, quantitative phenotypic and genotypic analyses in multicellular model organisms. Drosophila melanogaster is an indispensable model for studying the development and function of higher order organisms due to extensive genome homology, tractability, and its relatively short lifespan. Presently, nine Nobel prizes serve as a testament to the utility of this elegant model system. Ongoing advancements in genetic and molecular tools allow for the underlying mechanisms of human disease to be investigated in Drosophila. However, the absence of a method to precisely age-match tissues during larval development prevents further capitalization of this powerful model organism. Drosophila spends nearly half of its life cycle progressing through three morphologically distinct larval instar stages, during which the imaginal discs, precursors of mature adult external structures (e.g., eyes, legs, wings), grow and develop distinct cell fates. Other tissues, such as the central nervous system, undergo massive morphological changes during larval development. While these three larval stages and subsequent pupal stages have historically been identified based on the number of hours post egg-laying under standard laboratory conditions, a reproducible, efficient, and inexpensive method is required to accurately age-match larvae within the third instar. The third instar stage is of particular interest, as this developmental stage spans a 48-hr window during which larval tissues switch from proliferative to differentiation programs. Moreover, some genetic manipulations can lead to developmental delays, further compounding the need for precise age-matching between control and experimental samples. This article provides a protocol optimized for synchronous staging of Drosophila third instar larvae by colorimetric characterization and is useful for age-matching a variety of tissues for numerous downstream applications. We also provide a brief discussion of the technical challenges associated with successful application of this protocol. © 2023 Wiley Periodicals LLC. Basic Protocol: Synchronization of third instar Drosophila larvae.

Polycomb safeguards imaginal disc specification through control of the Vestigial-Scalloped complex.

A fundamental goal of developmental biology is to understand how cell and tissue fates are specified. The imaginal discs of Drosophila are excellent model systems for addressing this paradigm as their fate can be redirected when discs regenerate after injury or when key selector genes are misregulated. Here, we show that when Polycomb expression is reduced, the wing selector gene vestigial is ectopically activated. This leads to the inappropriate formation of the Vestigial-Scalloped complex, which forces the eye to transform into a wing. We further demonstrate that disrupting this complex does not simply block wing formation or restore eye development. Instead, immunohistochemistry and high-throughput genomic analysis show that the eye-antennal disc unexpectedly undergoes hyperplastic growth with multiple domains being organized into other imaginal discs and tissues. These findings provide insight into the complex developmental landscape that tissues must navigate before adopting their final fate.

TnaA, a trithorax group protein, modulates wingless expression in different regions of the Drosophila wing imaginal disc

wingless expression is exquisitely regulated by different factors and enhancers in the imaginal wing discs of Drosophila melanogaster in four domains: the dorsal band, the dorso-ventral boundary, and the inner and outer ring domains. tonalli is a trithorax group gene that encodes a putative SUMO E3 ligase that binds to chromatin to regulate the expression of its targets, including the Hox genes. However, its role in modulating gene expression is barely known. Here, we show that TnaA modulates the wingless expression at two domains of the wing disc, the dorso-ventral boundary and the inner ring. At first, tonalli interacts genetically with Notch to form the wing margin. In the inner ring domain, TnaA modulates wingless transcription. When the dosage of TnaA increases in or near the inner ring since early larval stages, this domain expands with a rapid increase in wingless expression. TnaA occupies the wingless Inner Ring Enhancer at the wing disc, meanwhile it does not affect wingless expression directed by the Ventral Disc Enhancer in leg discs, suggesting that TnaA acts as a wingless enhancer-specific factor. We describe for the first time the presence of TnaA at the Inner Ring Enhancer as a specific regulator of wingless in the development of wing boundaries.

TMED10 mediates the loading of neosynthesised Sonic Hedgehog in COPII vesicles for efficient secretion and signalling.

The morphogen Sonic Hedgehog (SHH) plays an important role in coordinating embryonic development. Short- and long-range SHH signalling occurs through a variety of membrane-associated and membrane-free forms. However, the molecular mechanisms that govern the early events of the trafficking of neosynthesised SHH in mammalian cells are still poorly understood. Here, we employed the retention using selective hooks (RUSH) system to show that newly-synthesised SHH is trafficked through the classical biosynthetic secretory pathway, using TMED10 as an endoplasmic reticulum (ER) cargo receptor for efficient ER-to-Golgi transport and Rab6 vesicles for Golgi-to-cell surface trafficking. TMED10 and SHH colocalized at ER exit sites (ERES), and TMED10 depletion significantly delays SHH loading onto ERES and subsequent exit leading to significant SHH release defects. Finally, we utilised the Drosophila wing imaginal disc model to demonstrate that the homologue of TMED10, Baiser (Bai), participates in Hedgehog (Hh) secretion and signalling in vivo. In conclusion, our work highlights the role of TMED10 in cargo-specific egress from the ER and sheds light on novel important partners of neosynthesised SHH secretion with potential impact on embryonic development.